The first vaccines consisted of live, attenuated pathogens. The attenuated forms were either naturally occurring closely related organisms or obtained through serial passages in culture. For example, tuberculosis (TB) in man has for many years been combated by vaccination with an attenuated strain of Mycobacterium bovis, —the M. bovis BCG vaccine developed more than 80 years ago. However, although more than 3 billion doses of BCG have been administered (more than any other vaccine) it does not always provide satisfactory resistance to human TB in every population.
Today, a more up-to-date approach is to use highly purified substances, e.g. purified recombinant proteins or peptides. These vaccines are well-defined and side-reactions are minimized. Unfortunately, many highly purified substances are not very immunogenic and do not induce a sufficient immune response to confer protection. To do this, the antigen needs some help from immune response potentiating agents called adjuvants. Depending on the pathogen, protection may require that either a humoral or a cell-mediated response predominates. An immune reaction that can be transferred with immune serum is termed humoral immunity and refers to resistance that is mediated by antibodies which bind to antigenic material associated with an infectious agent and thereby trigger an immune response against it. Cell-mediated immunity (CMI) relies on the cells of the immune system mounting an immune response. A CMI, or T helper (Th)1, immune response is generally associated with combating intracellular pathogens, including Leishmania, and Tuberculosis, but also has a role in combating other types of infection e.g. the yeast infection Candida. A humoral, or Th2, immune response is required for defence against extracellular pathogens e.g. helminth infections.
In a number of cases e.g. Influenza, Hepatitis C (HCV), Human Immunodeficiency Virus (HIV), Chlamydia and Malaria depending on the stage of infection, a mixed Th1/Th2 response may be required (Mosmann and Sad 1996). These require both Th1 and Th2 because parts of their lifecycle are intracellular but they also go through extracellular phases e.g. transmission between cells.
The development of a specific kind of immune response (humoral or cell-mediated) can be determined by the choice of adjuvant. For example, protective immunity against intracellular pathogens like M. tuberculosis requires a cell-mediated immune response, and a suitable adjuvant for a subunit vaccine directed against TB should enhance a Th1 response (Lindblad et al. 1997).
A large number of adjuvants exist but most of these suffer from numerous problems that preclude their use in humans. Only a few adjuvants are accepted for human use e.g. aluminum-based adjuvants (AlOH-salts) and MF-59, but they both induce Th2-biased responses, which makes them unsuitable for a TB vaccine and other vaccines requiring a Th1 response (Lindblad et al. 1997).
During the past 20-30 years a number of new adjuvant systems have been identified and some of those are currently under development. Despite this, the need for new adjuvant systems is still recognized (Moingeon et al. 2001) and is evident in the paucity of choices available for clinical use.
An adjuvant (from latin adjuvare, to help) can be defined as any substance that when administered in the vaccine serves to direct, accelerate, prolong and/or enhance the specific immune response. Adjuvants has been divided into two major categories either delivery systems or immunomodulators/immunostimulators. The delivery system can e.g. be emulsions, polystyrene particles, niosomes, ISCOMS, virosomes, microspheres, or surfactant-like liposomes, which are vesicles made up of lipid bilayers. The liposomes act as carriers of the antigen (either within the vesicles or attached onto the surface) and may form a depot at the site of inoculation allowing slow, continuous release of antigen. For some time after injection and phagocytosis, liposomal presentation ensures that a specific amount of antigen is made available to single antigen-presenting cells (Gluck 1995). The immunomodulators targets distinct cells or receptor e.g. toll-like receptors on the surface of APCs. Delivery systems and immunomodulators can be used together e.g. as in Glaxo's series of adjuvants. Therefore, in addition to delivering the vaccine antigen delivery system can also be used for delivering the immunomodulators.
In addition to being a component in a vaccine, immunomodulators can be administered without antigen(s). By this approach it is possible to activate the immune system locally e.g. seen as maturation of antigen-presenting cells, cytokine production which is important for anti-tumor and anti-viral activity. Thus, the administration of immunomodulators may e.g. support in the eradication of cancer and skin diseases. Examples of immunomodulators which can be administered locally are Taxanes e.g. Taxol, the toll-like receptor 7/8 ligand Resiquimod, Imiquimod, Gardiquimod.
Dimethyldioctadecylammonium-bromide, -chloride, -phosphate, -acetate or other organic or inorganic salts (DDA) is a lipophilic quaternary ammonium compound, which forms cationic liposomes in aqueous solutions at temperatures above 40° C. DDA is a very efficient delivery system enhancing the uptake of vaccine antigen into APCs. Combinations of DDA and immunomodulating agents have been described. Administration of Arquad 2HT, which comprises DDA, in humans was promising and did not induce apparent side effects (Stanfield, 1973). The combination of DDA and TDB or DDA and MPL showed a very clear synergy between the delivery vehicle (DDA) and the immunomodulator (TDB or MPL) with highly elevated levels of CMI response compared to the response obtained with either components alone. DDA is therefore a promising delivery vehicle for vaccine antigen and an immunomodulator e.g. in the development of an adjuvant system for a vaccine against TB and other intracellular pathogens.
Various compounds from mycobacteria have been reported to be immunomodulating. When lipids extracted from M. bovis BCG were used as an adjuvant, a skin test response to ovalbumin was obtained in guinea pigs (Hiu 1975). Liposomes formed at elevated temperatures from total polar lipids of M. bovis BCG are able to generate a humoral response to ovalbumin, and a vaccine prepared from these polar lipids gave protection in mice upon challenge with tumor cells (WO 03/011336). The effect of total lipids from M. tuberculosis H37Rv as antigen in an experimental TB vaccine for guinea pigs was investigated by (Dascher et al. 2003). In this study, liposomes based on cholesterol and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) were mixed with a M. tuberculosis H37Rv total lipid extract. After removing the solvent, the lipids were reconstituted with dimethyldioctadecylammonium (DDA) as an adjuvant in PBS buffer. Guinea pigs immunised with this vaccine did not show a significant reduction in bacteria, suggesting that this formulation of liposomes mixed with DDA lacks a strong antigen or that the formulation of mycobacterial lipids with Cholesterol:DSPC prevent the adjuvanting effect of DDA. Alternatively, as a mixture of various lipids is administered, the effective lipids may constitute too limited a proportion of the total lipids.
Various purified components from mycobacteria have also been investigated for their adjuvant activity. Purified protein derivative (PPD) did not induce a delayed type hypersensitivity reaction on its own, but when Wax D (a mycobacterial cell wall peptidoglycan fragment-arabinogalactan-mycolic acid complex) was added as an adjuvant, a strong reaction was observed (Yamazaki S 1969). The immunomodulator SSM or Z-100, a lipid arabinomannan extracted from M. tuberculosis, has antitumor activity (Suzuki F 1986). Another mycobacterial cell-derived compound is trehalose 6,6′-dimycolate (TDM) (cord factor; a mycolic acid containing glycolipid) (Saito et al, 1976). Also, TDM (or synthetic analogues) has immunostimulatory effects and has been included in various adjuvant formulations (McBride et al. 1998) (Koike et al. 1998).
In a paper by Silva et al (1985), five components purified fra Mycobacterium bovis BCG was injected intravenously as lipid-coated charcoal particles and gave rise to an inflammatory reaction in the lungs of mice. The five components included TDM, trehalose monomycolate (MMT), glucose monomycolate (MMGlc), arabinose monomycolate (MMAr), and a glycerol monomycolate (MMGlyc). The paper describes the monomycolylglycerol headgroup whereas the composition of the mycolid acids is poorly defined and no structural data are provided. In addition, the reaction upon administration of the lipids is only described as an inflammatory activity in the lungs whereas the ability to enhance a specific immune response known as an adjuvant effect is not described.
Although the immunostimulatory and inflammatory activity of mycobacterial-derived lipids has been recognised for many decades with an ever-expanding literature of lipids capable of stimulating immune responses in animal (murine) models, to date individual lipid(s) with the ability to stimulate human dendritic cells (DCs) have not been identified. In example; although TDM has shown to be the most active mycobacterial lipid in terms of proinflammatory responses, no activation of dendritic cells upon stimulation with TDM has been observed (Uehori et al, 2003). So although TDM has shown inflammatory activity in several papers, this lipid apparently lacks the ability to activate the dendritic cells that are crucial for initiating an immune response. The identification of such a lipid with the ability to activate human dendritic cells would suggest that it could be used as part of a novel adjuvant system that would be suitable for use in humans. Furthermore, the lack of Th1-inducing adjuvants suitable for human use makes the identification of a single, mycobacteria-derived lipid with Th1-promoting capability a significant finding.
DCs are professional antigen presenting cells (APC) that play an essential role in directing the immune response upon infection with pathogens, such as M. tuberculosis. Hence, the production of IL-12 by activated DC represents a vital step in controlling M. tuberculosis infection since it is this cytokine that is of paramount importance in driving the production of IFN-γ by Th-1 cells, which promotes the activation of macrophages (Nathan et al. 1983). Furthermore, in recent years evidence has been uncovered that indicates that mycobacteria also target DC in an attempt to modulate the immune response and a crucial role for mycobacterial lipids in this process has been established.
Up to 40% of the dry weight of the cell envelope of mycobacteria is comprised of lipids (Minnikin 1982). These lipids have long been associated with the distinctive pathogenicity of this family of organisms and are known to play a substantial role in the host response to mycobacterial infection (Brennan and Goren 1979). Prominent among these lipids are the phthiocerol dimycocerosate (PDIM) waxes (Minnikin et al. 2002) the presence of which has been shown to correlate with pathogenicity; PDIM-deficient M. tuberculosis mutants show attenuated growth in mice (Sirakova et al. 2003). Closely related to the PDIMs are the so-called “phenolic glycolipids” (PGLs), a good example being the 2-methylrhamnosyl phenolphthiocerol dimycocerosates (“mycoside B”) found in Mycobacterium bovis. A link between this monoglycosyl PGL and the hypervirulence of certain isolates of M. tuberculosis has recently been demonstrated (Reed et al. 2004).
Another lipid class of particular interest are the trehalose-6,6-dimycolates (TDM), the so-called “cord factors”. TDM promotes the maintenance of granulomatous lesions by stimulating the release of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-12, and the Th-1-promoting cytokine IFN-γ (Lima et al. 2001) and has a role in prolonging the survival of M. tuberculosis inside macrophages by inhibiting phagosome-lysosome fusion (Indrigo et al. 2002). The fine structure of the mycolate components of TDM is important in the proinflammatory activation of macrophages during early infection (Rao et al, 2005).
Despite their role in enhancing mycobacterial survival, the immunomodulatory powers of mycobacterial lipids can also be harnessed to create a new generation of Th1-inducing adjuvants. In identifying individual lipids with potent immunostimulatory activity, it may be possible to circumvent the problems with toxicity associated with the use of heat-killed whole cells of M. tuberculosis mixed with oil—Freund's complete adjuvant (CFA)—while still maintaining the potent adjuvant activity. Indeed, liposomes formed from the polar lipids of M. bovis Bacillus-Calmette-Guerin (BCG) have recently been shown to activate murine bone marrow-derived dendritic cells (BM-DC). The majority of this activity was found to be attributable to the lipid glycophospholipid phosphatidylinositol dimannoside (Sprott et al. 2004).
Recent studies in our laboratories have characterised the novel adjuvant combination of a mycobacterial lipid extract from M. bovis BCG and dimethyldioctadecylammonium bromide (DDA) that is capable of promoting a complex and sustained immune response, with both a strong humoral and cell-mediated component (Rosenkrands et al. 2005). The majority of the adjuvant activity of the total BCG lipids was found to be attributable to the apolar lipids.
While these studies further confirm the potential of mycobacterial lipids to act as adjuvants, the optimal solution would be to identify the single most immunostimulatory lipid which, alone, has potent activity. This would represent an even simpler, cheaper adjuvant and would also raise the possibility of making synthetic analogues of the lipid allowing for a cleaner system that could be produced in the large quantities required of an adjuvant for use in vaccines that are administered worldwide.